This project will determine how the presence of disease-linked microtubule associating proteins (MAPs) influence and regulate microtubule (MT) structure. In particular, we will investigate two important MAPs: tau and EB1. Normal tau binds to and regulates the growth and stability of MTs while certain tau mutations are known to cause neurological disease. EB1 binds to the growing plus-end of MTs, coordinates other end binding proteins and possibly changes the lattice structure of MTs. A wide array of cancer cells has been found to over- express EB1. Using a custom-built multimodal microscope capable of simultaneous single-molecule fluorescence and optical trapping, this project will determine the effects of MAPs on MT stiffness and assess the ability of MAP-coated MTs to support kinesin-based transport. These investigations will first consider tau mutations known to cause frontotemporal dementia with parkinsonism-17, and progressive supranuclear palsy, devastating neurological disorders. The hypothesis that the GTP-rich EB1-coated plus end of a MT has differing stiffness and supports different rates of kinesin translocation compared to the rest of the MT will also be tested. A passive fluctuation assay and an active bending assay will be used to measure flexibility changes in microtubules under small and large deformations. Nanometer resolution single- molecule optical trapping experiments enable measurements of not only translocation speed, but also step size, dwell time, run length, forward to backward stepping ratio, etc. The force dependence of these parameters will be characterized as well. These complimentary data sets will provide critical quantitative insight into the role of MT mechanics and structure in health and disease. In the short term, this project will answer fundamental biological questions about the role of MAPs in regulating the organization and function of the MT cytoskeleton. Ultimately, answers to these questions will contribute to improved medical diagnostics and/or treatments of diseases linked to MT and MAP dysfunction, including neuropathies, Alzheimer's disease and cancer.